Phosphoenolpyruvate Carboxykinase (GTP) Gene Transcription and Hyperglycemia Are Regulated by Glucocorticoids in Genetically Obesedb/db Transgenic Mice

Jacob E Friedman,Yang Sun,Tatsuya Ishizuka,Craig J Farrell,Shana E Mccormack,Lisa M Herron,Parvin Hakimi,Pamela Lechner,Jeung S Yun

doi:10.1074/jbc.272.50.31475

Abstract

The molecular mechanisms underlying increased hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene transcription and gluconeogenesis in type II diabetes are largely unknown. To examine the involvement of glucocorticoids and the cis-acting insulin response sequence (IRS, -416/-407) in the genetically obese db/db mouse model, we generated crosses between C57BL/KsJ-db/+ mice and transgenic mice that express -460 or -2000 base pairs of the rat PEPCK gene promoter containing an intact or mutated IRS, linked to a reporter gene. Transgenic mice expressing the intact PEPCK(460)-CRP (C-reactive protein) transgene bred to near homozygosity at the db locus were obese, hyperinsulinemic, and developed fasting hyperglycemia (389 +/- 26 mg/100 ml) between 4 and 10 weeks of age. Levels of CRP reporter gene expression were increased 2-fold despite severe hyperinsulinemia compared with non-diabetic non-obese transgenic mice. Reporter gene expression was also increased 2-fold in transgenic obese diabetic db/db mice bearing a mutation in the IRS, -2000(IRS)-hGx, compared with non-obese non-diabetic transgenic 2000(IRS)-hGx mice. Treatment of obese diabetic db/db transgenic mice with the glucocorticoid receptor blocker RU 486 decreased plasma glucose by 50% and reduced PEPCK, GLUT2, glucose-6-phosphatase, tyrosine aminotransferase, CRP, and hGx reporter gene expression to levels similar to those of non-obese normoglycemic transgenic mice. Taken together, these results establish that -460 bp of 5'-flanking sequence is sufficient to mediate the induction of PEPCK gene transcription in genetically obese db/db mice during the development of hyperglycemia. The results further demonstrate that the mechanism underlying increased expression of gluconeogenic enzymes in the db/db mouse requires the action of glucocorticoids and occurs independently of factors acting through the PEPCK IRS (-416/-407) promoter binding site.

Highlights

Introduction of phosphoenolpyruvate carboxykinase (PEPCK) Transgene intoC57BL/KsJ-db/ϩ Mice—To study the effects of obesity and type II diabetes on promoter function, mice homozygous for the autosomal recessive db gene containing either intact or mutated promoter were produced using the breeding procedure outlined previously for outcrossing db/db mice [26]
The present results suggest that elements located in the proximal Ϫ460 bp of the PEPCK promoter are of major importance in up-regulating PEPCK gene expression in the hyperinsulinemic obese diabetic db/db mouse
The elevated plasma C-reactive protein gene (CRP) levels observed during the onset of hyperglycemia in transgenic PEPCK[460]-CRP, db/db mice and the fact that the half-life for clearance of circulating rabbit CRP in transgenic mice is 45 min [18] suggest a shift in hepatic metabolism during the postweaning stage and increases in transcription from the PEPCK gene promoter

Summary

EXPERIMENTAL PROCEDURES

Materials—ATP, CTP, GTP, yeast tRNA, proteinase K, and restriction enzymes were purchased from Boehringer Mannheim. [␣-32P]dCTP (3000 Ci/mmol), [␣-32P]UTP (3000 Ci/mmol), and GeneScreen Plus were purchased from NEN Life Science Products. The first utilized homozygous transgenic C57B6/SJL mice (a hybrid of C57BLK ϫ SJL) containing sequences from Ϫ460 to ϩ73 bp of the rat cytosolic PEPCK gene ligated to the rabbit CRP gene, PEPCK[460]CRP, obtained from previously established transgenic lines Transgenic mice were identified by Southern blot analysis, using a random primed 32P-labeled 2.2-kb BamHI-EcoRV hGx gene fragment to probe samples of DNA isolated from mouse tails [25]. Introduction of PEPCK Transgene into C57BL/KsJ-db/ϩ Mice—To study the effects of obesity and type II diabetes on promoter function, mice homozygous for the autosomal recessive db gene containing either intact or mutated promoter were produced using the breeding procedure outlined previously for outcrossing db/db mice [26].

CRP n

RESULTS

DISCUSSION